Metal-ion-dependent folding of a uranyl-specific DNAzyme: insight into function from fluorescence resonance energy transfer studies.

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO(2)(2+))-specific 39E DNAzyme in the presence of Mg(2+), Zn(2+), Pb(2+), or UO(2)(2+). At pH 5.5 and physiological ionic strength (100 mM Na(+)), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg(2+) or Zn(2+). However, no folding occurred in the presence of Pb(2+) or UO(2)(2+); this is analogous to the "lock-and-key" catalysis mode first observed in the Pb(2+)-specific 8-17 DNAzyme. However, Mg(2+) and Zn(2+) exert different effects on the 8-17 and 39E DNAzymes. Whereas Mg(2+) or Zn(2+)-dependent folding promoted 8-17 DNAzyme activity, the 39E DNAzyme folding induced by Mg(2+) or Zn(2+) inhibited UO(2)(2+)-specific activity. Group IIA series of metal ions (Mg(2+), Ca(2+), Sr(2+)) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr(2+) (1.12 Å) is comparable to that of Pb(2+) (1.20 Å), but contrary to Pb(2+), Sr(2+) induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb(2+) and UO(2)(2+). Under low ionic strength (30 mM Na(+)), all four metal ions (Mg(2+), Zn(2+), Pb(2+), and UO(2)(2+)), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA-backbone phosphates in addition to playing specific catalytic roles. Mg(2+) at low (<2 mM) concentration promoted UO(2)(2+)-specific activity, whereas Mg(2+) at high (>2 mM) concentration inhibited the UO(2)(2+)-specific activity. Therefore, the lock-and-key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock-and-key mode only at ionic strengths of 100 mM or greater.